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20 Maciej Gagat et al. W y n i k i . W wyniku ekspozycji komórek HL-60 na podwyższoną temperaturę obserwowano reorganizację cytoszkieletu aktynowego oraz pojawienie się komórek o cechach charakterystycznych dla procesu apoptozy, takich jak obkurcznie cyto- i nukleoplazmy, kondensacja i marginalizacja chromatyny czy obrzmienie mitochondriów. W wyniku rearanżacji, F-aktyna lokalizowała sie głównie w części korowej komórki w formie pierścienia lub w cytoplazmie w postaci wyraźnie wyznakowanych sieci i skupień. Stopień nasilenia zmian w komórkach wzrastał wraz ze wzrostem czasu regeneracji komórek. W n i o s k i . Uzyskane wyniki pozwalają stwierdzić, że cytoszkielet aktynowy zaangażowany jest w realizację procesu apoptozy indukowanej przez łagodną hipertermię. Ponadto sugerują one, że na wystąpienie zmian w organizacji filamentów aktynowych, jak również zmian w morfologii i ultrastrukturze komórek ma wpływ, nie tylko zastosowany profil temperaturowy, ale również czas regeneracji komórek. Key words: hyperthermia, actin filaments, HL-60 cell line Słowa kluczowe: hipertermia, filamenty aktynowe, linia komórkowa HL-60 INTRODUCTION The term ‘hyperthermia’ refers to raising the temperature of a part of the body or of the whole body above the threshold temperature set at a particular moment by the thermoregulatory system of the organism [1,2]. Hyperthermia has been used for its medical properties since ancient times. Presumably, the oldest written medical report with references to hyperthermia was found in the Egyptian Edwin Smith surgical papyrus, dated around 3000 BC [3]; whereas, the use of hyperthermia for cancer therapy was first reported by Hypocrites in the treatment of breast tumor [4]. Currently, hyperthermia is a well-established physical modality, which is used as an adjunctive therapy with various established cancer treatments, such as radiotherapy and chemotherapy [5]. The scientific rationales for using hyperthermia in cancer treatment are based on the data from in vitro, animal and preclinical studies [3,6]. It has been shown that the alterations in plasma membrane permeability caused by hyperthermia lead to better infiltration and drug absorption by the tumor [7]. It has been also suggested that hyperthermia treatment activates the immune system against the tumor [8]. Moreover, cancer cells are selectively sensitive to heat shock treatment, while the same conditions rarely affect the growth of normal cells. Additionally, an increase in thermosensitivity of tumor cells results in the reduction of tumor blood flow which makes their environment more hypoxic and acidic. Moreover, several studies have shown that hypoxia and particularly acidity enhance the cytotoxicity of hyperthermia [9,10]. The mechanisms of hyperthermia cytotoxicity involve denaturation of cellular proteins and their subsequent aggregation as well as plasma membrane damage, inhibition of DNA repair, and changes in intracellular calcium homeostasis. The heat-induced alterations in cell structure and function lead to cell cycle arrest and cell death [11]. Among the most noteworthy alterations in heatshocked cells are the induction of heat shock proteins (HSPs) and reorganization of the cytoskeleton [12]. The actin cytoskeleton is involved in the regulation of fundamental cellular processes such as motility, cytokinesis, adhesion and endocytosis [13]. Furthermore, it also participates in signal transduction regulating cell growth, survival, and death. Therefore, the actin cytoskeleton may represent an attractive target for hyperthermic therapy against different types of cancer [13, 14]. However, the mechanism of structural and functional actin remodeling in response to heat shock is not fully understood. The aim of this study was to determine the effect of mild hyperthermia on actin cytoskeleton organization in the HL-60 cell line, in the context of involvement in cell death process. MATHERIAL AND METHODS Cell culture and treatment The human promyelocytic leukemia cell line HL-60 was purchased from the American Type Culture Collection (ATCC, Manassas, VA; CCL-240). The cells were cultured in RPMI 1640 medium (Lonza) supplemented with 10% fetal bovine serum (FBS, PAA) and 10 mg/ml gentamicin (Lonza), at 37ºC in a humidified 5% CO 2 atmosphere. After 48 hr cell culture, HL-60 cells were subjected to heat shock (40ºC, 2 hrs), followed by post-heat shock recovery at 37ºC for 0, 3 and 6 hrs. Control cells were cultured identically without exposure to heat treatment. Cell viability was assessed by the trypan blue dye exclusion method.
The effect of mild hyperthermia on morphology, ultrastructure and F-actin organization in HL-60 cell line 21 Light microscopy studies For the morphological analysis, HL-60 cells were fixed in 4% paraformaldehyde. After fixation, the cells were incubated with 0.1M glycine solution (Roth) and then the cell suspension was centrifuged onto glass slides. Thereafter, the cells were stained with Mayer's hematoxylin and rinsed under running tap water and dehydrated in a graded series of alcohols and xylenes. The preparations were observed using an Eclipse E800 microscope (Nikon) with NIS-Elements ver. 3.30 image analysis system (Nikon) and CCD camera (DS-5Mc-U1; Nikon). Transmission electron microscopy studies For ultrastructural analysis, the cells were fixed with 3.6% glutaraldehyde and then postfixed with 1% osmium tetroxide, dehydrated with graded series of alcohol and acetone, and embedded in Epon 812. The polymerization of the resin was performed at 37°C for 24 hrs, and then at 65°C for 120 hrs. Selected parts of material were cut into ultra-thin sections by using an OmU3 ultramicrotome (Reichert) and then counterstained with uranyl acetate and lead citrate. The material was examined using JEM 100 CX electron microscope (JEOL). Fluorescence microscopy studies For fluorescence labeling of actin, the cells were fixed with 4% paraformaldehyde. After fixation, the cells were incubated with 0.1M glycine solution (Roth) and then the cell suspension was centrifuged onto glass slides. The cells were then permeabilized with 0.1% Triton X-100 (Serva Feinbiochemica). To enable visualization of F-actin, the cells were incubated with phalloidin conjugated to Alexa Fluor 488 (Invitrogen, diluted 1:40). The nuclei of the cells were labeled with 4’,6-diamidino-2-phenyloindole (DAPI; Sigma- Aldrich). Slides were mounted in Aqua-Poly/Mount (Polysciences) and analysed by using an Eclipse E800 microscope with a Y-FL fluorescence attachment (Nikon), NIS-Elements ver. 3.30 image analysis system (Nikon) and CCD camera (DS-5Mc-U1; Nikon). Statistical analysis The non-parametric Mann-Whitney U test was performed to compare the differences between experimental groups. The results were considered statistically significant at p
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